Welcome to Acta Agronomica Sinica,

Acta Agron Sin ›› 2016, Vol. 42 ›› Issue (03): 389-398.doi: 10.3724/SP.J.1006.2016.00389

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Cloning of 6-SFT Gene from Leymus racemosus and Analysis of Tolerance to Drought and Cold Stresses in Transgenic Tobacco

HE Xiao-Lan1,WANG Jian-Wei2,LI Wen-Xu3,CHEN Zhen-Zhen1,ZHAO Ji-Xin1,WU Jun1,WANG Zhong-Hua1,CHEN Xin-Hong1,*   

  1. 1 Shaanxi Provincial Key Laboratory of Plant Genetic Engineering Breeding / College of Agronomy, Northwest A&F University, Yangling 712100, China; 2 College of Environment and Life Science, Kaili University, Kaili 556011, China; 3 Wheat Research Institute, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China
  • Received:2015-07-22 Revised:2015-11-20 Online:2016-03-12 Published:2015-12-18
  • Contact: 陈新宏, E-mail: cxh2089@126.com; Tel: 029-87082854 E-mail:helingzhi123@126.com
  • Supported by:

    This study was supported by the National Natural Science Foundation of China (31571650) and the Tang Zhong-Ying Breeding Funding Project of the Northwest A&F University.

Abstract:

The fructan biosynthesis enzyme (6-SFT) plays an important role in plant responses to abiotic stresses. In this study, a full-length cDNA encoding sucrose:fructan-6-fructosyltransferase, designated as Lr-6-SFT (GenBank accession No. KT387273), was cloned from Leymus racemosus (2n = 4x = 28, NsNsXmXm) using reverse transcriptase PCR (RT-PCR) and rapid-amplification of cDNA ends (RACE) techniques. The full-length open reading frame comprises 1863 bp and encodes 620 amino acids. The predicted protein structure of the gene containes a conserved fructosyltransferase domain. Multiple sequence alignment and phylogenetic analysis showed that the Lr-6-SFT protein shared high similarity with 6-SFT proteins from Psathyrostachys huashanica, Triticum aestivum, Aegilops searsii, and Hordeum vulgare subsp. vulgare. The Lr-6-SFT gene was transferred into tobacco (Nicotiana tabacum L. cv. W38) via Agrobacterium-mediated transformation. The screened plants were tested by PCR and RT-PCR, and the transgenic tobacco plants exhibited much higher tolerance to drought and cold compared with the non-transgenic plants. Under drought and cold stresses, the Lr-6-SFT expressions were associated with the increased accumulation of stored carbohydrate and proline and the decreased malondialdehyde storage. These results suggest that Lr-6-SFT is a typical member of the glycoside hydrolase 32 (GH32) family and may be linked to enhanced tolerance to drought and cold stresses.

Key words: 6-SFT gene, Drought, Freezing, Leymus racemosus, Physiological indices, Transgenic tobacco

[1] Van Arkel J, Sévenier R, Hakkert J C, Bouwmeester H J, Koops A J, van der Meer I M. Tailor-made fructan synthesis in plants: a review. Carbohydr Polym, 2013, 93: 48–56

 [2] Hendry G A, Wallace R K. The origin, distribution, and evolutionary significance of fructans. In: Suzuki M, Chatterton N J, eds. Science and Technology of Fructans, Boca Raton, Florida, USA: The Chemical Rubber Company Press, 1993. pp 119–139

 [3] Wiemken A, Frehner M, Keller F, Wagner W. Fructan metabolism, enzymology and compartmentation: current topics in plant biochemistry and physiology. In: Proceedings of the Plant Biochemistry and Physiology Symposium. Columbia, USA: University of Missouri, 1986. pp 17–37

 [4] Hincha D K, Livingston D R, Premakumar R, Zuther E, Obel N, Cacela C, Heyer A G. Fructans from oat and rye: composition and effects on membrane stability during drying. Bioch Biophys Acta, 2007, 1768: 1611–1619

 [5] Bie X M, Wang K, She M Y, Du L, Zhang S X, Li J R, Gao X, Lin Z S, Ye X G. Combinational transformation of three wheat genes encoding fructan biosynthesis enzymes confers increased fructan content and tolerance to abiotic stresses in tobacco. Plant Cell Rep, 2012, 31: 2229–2238

 [6] Ritsema T, Joling J, Smeekens S. Patterns of fructan synthesized by onion fructan:fructan 6G-fructosyltransferase expressed in tobacco BY2 cells-is fructan:fructan 1-fructosyltransferase needed in onion? New Phytol, 2003, 160: 61–67

 [7] Sprenger N, Bortlik K, Brandt A, Boller T, Wiemken A. Purification, cloning, and functional expression of sucrose:fructan 6-fructosyltransferase, a key enzyme of fructan synthesis in barley. Proc Natl Acad Sci USA, 1995, 92: 11652–11656

 [8] Wei J Z, Chatterton N J. Fructan biosynthesis and fructosyltransferase evolution: expression of the 6-SFT (sucrose:fructan 6-fructosyltransferase) gene in crested wheatgrass (Agropyron cristatum). J Plant Physiol, 2001, 158: 1203–1213

 [9] Kawakami A, Yoshida M. Molecular characterization of sucrose:sucrose 1-fructosyltransferase and sucrose:fructan 6-fructosyltransferase associated with fructan accumulation in winter wheat during cold hardening. Biosci Biotech Biochem, 2002, 66: 2297–2305

[10] Tamura K I, Kawakami A, Sanada Y, Tase K, Komatsu T, Yoshida M. Cloning and functional analysis of a fructosyltransferase cDNA for synthesis of highly polymerized levans in timothy (Phleum pratense L.). J Exp Bot, 2009, 60: 893–905

[11] Del Viso F, Puebla A F, Fusari C M, Casabuono A C, Couto A S, Pontis H G, Hopp H E, Heinz R A. Molecular characterization of a putative sucrose:fructan 6-fructosyltransferase (6-SFT) of the cold-resistant Patagonian grass Bromus pictus associated with fructan accumulation under low temperatures. Plant & Cell Physiol, 2009, 50: 489–503

[12] He X L, Chen Z Z, Wang J W, Li W X, Zhao J X, Wu J, Wang Z H, Chen X H. A sucrose:fructan-6-fructosyltransferase (6-SFT) gene from Psathyrostachys huashanica confers abiotic stress tolerance in tobacco. Gene, 2015, 570: 239–247

[13] 高翔. 小麦族植物果聚糖合成酶基因克隆及功能验证. 中国农业科学院博士学位论文, 北京, 2010

Gao X. Cloning and Functional Analysis of Genes Encoding Fructan Biosynthesis Enzymes in Triticeae Plants. PhD Dissertation of Chinese Academy of Agricultural Sciences, Beijing, China, 2010 (in Chinese with English abstract)

[14] Del Viso F, Casabuono A C, Couto A S, Hopp H E, Puebla A F, Heinz R A. Functional characterization of a sucrose:fructan 6-fructosyltransferase of the cold-resistant grass Bromus pictus by heterelogous expression in Pichia pastoris and Nicotiana tabacum and its involvement in freezing tolerance. J Plant Physiol, 2011, 168: 493–499

[15] Li H J, Yang A F, Zhang X C, Gao F, Zhang J R. Improving freezing tolerance of transgenic tobacco expressing sucrose:sucrose 1-fructosyltransferase gene from Lactuca sativa. Plant Cell Tissue Organ Cult, 2007, 89: 37–48

[16] Wei J Z, Jerry Chatterton N, Larson S R. Expression of sucrose:fructan 6-fructosyltransferase (6-SFT) and myoinositol 1-phosphate synthase (MIPS) genes in barley (Hordeum vulgare) leaves. J Plant Physiol, 2001, 158: 635-643

[17] Tamura K, Sanada Y, Tase K, Kawakami A, Yoshida M, Yamada T. Comparative study of transgenic Brachypodium distachyon expressing sucrose:fructan 6-fructosyltransferases from wheat and timothy grass with different enzymatic properties. Planta, 2014, 239: 783–792

[18] Knipp G, Honermeier B. Effect of water stress on proline accumulation of genetically modified potatoes (Solanum tuberosum L.) generating fructans. J Plant Physiol, 2006, 163: 392–397

[19] Ruelland E, Vaultier M, Zachowski A, Hurry V. Chapter 2 cold signalling and cold acclimation in plants. Adv Bot Res, 2009, 49: 35–150

[20] Szabados L, Savouré A. Proline: a multifunctional amino acid. Trends Plant Sci, 2010, 15: 89–97

[21] Schroeven L, Lammens W, Kawakami A, Yoshida M, Van Laere A, Van den Ende W. Creating S-type characteristics in the F-type enzyme fructan:fructan 1-fructosyltransferase of Triticum aestivum L. J Exp Bot, 2009, 60: 3687–3696

[22] Chatterton N J, Harrison P A, Bennett J H, Asay K H. Carbohydrate partitioning in 185 accessions of Gramineae grown under warm and cool temperatures. J Plant Physiol, 1989, 134: 169–179

[23] Chatterton N J, Harrison P A, Thornley W R, Bennett J H. Structure of fructan oligomers in cheatgrass (Bromus tectorum L.). New Phytol, 1993, 124: 389–396

[24] Mcguire P E, Dvorak J. High salt-tolerance potential in wheatgrasses. Crop Sci, 1981, 21: 702–705

[25] Pilon-Smits E, Ebskamp M, Paul M J, Jeuken M, Weisbeek P J, Smeekens S. Improved performance of transgenic fructan-accumulating tobacco under drought stress. Plant Physiol, 1995, 107: 125–130

[26] Heyer A G, Wendenburg R. Gene cloning and functional characterization by heterologous expression of the fructosyltransferase of Aspergillus sydowi IAM 2544. Appl Environ Microbiol, 2001, 67: 363–370

[27] Chen H, Nelson R S, Sherwood J L. Enhanced recovery of transformants of Agrobacterium tumefaciens after freeze-thaw transformation and drug selection. Biotechniques, 1994, 16: 664–668

[28] Schuler M A, Zielinski R E. Transformation of leaf discs with Agrobacterium. In: Gelvin S B, Schilperoort R A, Verma D P S, eds. Methods in Plant Molecular Biology. San Diego: Academic Press, 1989. pp 145–156

[29] Sambrook J, Fritsch E F, Maniatis T. Molecular Cloning (Third Edn). New York: Cold Spring Harbor Laboratory Press, 1989

[30] Fales F W. The assimilation and degradation of carbohydrates by yeast cells. J Biol Chem, 1951, 193: 113–124

[31] Bates L S, Waldren R P, Teare I D. Rapid determination of free proline for water-stress studies. Plant and Soil, 1973, 39: 205–207

[32] Lasseur B, Schroeven L, Lammens W, Le Roy K, Spangenberg G, Manduzio H, Vergauwen R, Lothier J, Prud' Homme M P, Van den Ende W. Transforming a fructan:fructan 6G-fructosyltransferase from perennial ryegrass into a sucrose:sucrose 1-fructosyltransferase. Plant Physiol, 2009, 149: 327–339

[33] 岳爱琴, 李昂, 毛新国, 昌小平, 李润植, 贾继增, 景蕊莲. 小麦果聚糖合成酶基因6-SFT-A单核苷酸多态性分析及其定位. 中国农业科学, 2011, 44: 2216–2224

Yue A Q, Li A, Mao X G, Chang X P, Li R Z, Jia J Z, Jing R L. Single nucleotide polymorphism and mapping of 6-SFT-A gene responsible for fructan biosynthesis in common wheat. Sci Agric Sin, 2011, 44: 2216–2224 (in Chinese with English abstract)

[34] Gao X, She M Y, Yin G X, Yu Y, Qiao W H, Du L P, Ye X G. Cloning and characterization of genes coding for fructan biosynthesis enzymes (FBEs) in triticeae plants. Agric Sci China, 2010, 9: 313–324

[35] 李淑洁, 李静雯, 张正英. Ta6-SFT在烟草中的逆境诱导型表达及抗旱性. 作物学报, 2014, 40: 994–1001

Li S J,Li J W,Zhang Z Y. Expression of Ta6-SFT Gene in tobacco induced by drought stress. Acta Agron Sin, 2014, 40: 994–1001 (in Chinese with English abstract)

[36] 文明, 卜利伟, 罗紫韵, 王佳伟, 董芬, 黄雪松. 大蒜蔗糖﹕蔗糖1-果糖基转移酶(1-SST)的酶学特征. 中国农业科学, 2015, 48: 334–342

Wen M, Bu L W, Luo Z Y, Wang J W, Dong F, Huang X S. Characteristics of sucrose:sucrose 1-fructosyltransferase in garlic. Sci Agric Sin, 2015, 48: 334–342 (in Chinese with English abstract)

[37] Tamura K, Sanada Y, Tase K, Yoshida M. Fructan metabolism and expression of genes coding fructan metabolic enzymes during cold acclimation and overwintering in timothy (Phleum pratense). J Plant Physiol, 2014, 171: 951–958

[38] 许欢欢, 康健, 梁明祥. 植物果聚糖的代谢途径及其在植物抗逆中的功能研究进展. 植物学报, 2014, 49: 209–220

Xu H H, Kang J, Liang M X. Research advances in the metabolism of fructan in plant stress resistance. Chin Bull Bot, 2014, 49: 209–220 (in Chinese with English abstract)

[39] Venkateswarlu B, Shanker A K, Shanker C, Maheswari M. Crop Stress and its Management: Perspectives and Strategies. Springer Netherlands, 2012

[1] CHEN Song-Yu, DING Yi-Juan, SUN Jun-Ming, HUANG Deng-Wen, YANG Nan, DAI Yu-Han, WAN Hua-Fang, QIAN Wei. Genome-wide identification of BnCNGC and the gene expression analysis in Brassica napus challenged with Sclerotinia sclerotiorum and PEG-simulated drought [J]. Acta Agronomica Sinica, 2022, 48(6): 1357-1371.
[2] ZHOU Wen-Qi, QIANG Xiao-Xia, WANG Sen, JIANG Jing-Wen, WEI Wan-Rong. Mechanism of drought and salt tolerance of OsLPL2/PIR gene in rice [J]. Acta Agronomica Sinica, 2022, 48(6): 1401-1415.
[3] LI Yi-Jun, LYU Hou-Quan. Effect of agricultural meteorological disasters on the production corn in the Northeast China [J]. Acta Agronomica Sinica, 2022, 48(6): 1537-1545.
[4] WANG Xing-Rong, LI Yue, ZHANG Yan-Jun, LI Yong-Sheng, WANG Jun-Cheng, XU Yin-Ping, QI Xu-Sheng. Drought resistance identification and drought resistance indexes screening of Tibetan hulless barley resources at adult stage [J]. Acta Agronomica Sinica, 2022, 48(5): 1279-1287.
[5] LI A-Li, FENG Ya-Nan, LI Ping, ZHANG Dong-Sheng, ZONG Yu-Zheng, LIN Wen, HAO Xing-Yu. Transcriptome analysis of leaves responses to elevated CO2 concentration, drought and interaction conditions in soybean [Glycine max (Linn.) Merr.] [J]. Acta Agronomica Sinica, 2022, 48(5): 1103-1118.
[6] WANG Xia, YIN Xiao-Yu, Yu Xiao-Ming, LIU Xiao-Dan. Effects of drought hardening on contemporary expression of drought stress memory genes and DNA methylation in promoter of B73 inbred progeny [J]. Acta Agronomica Sinica, 2022, 48(5): 1191-1198.
[7] DING Hong, XU Yang, ZHANG Guan-Chu, QIN Fei-Fei, DAI Liang-Xiang, ZHANG Zhi-Meng. Effects of drought at different growth stages and nitrogen application on nitrogen absorption and utilization in peanut [J]. Acta Agronomica Sinica, 2022, 48(3): 695-703.
[8] ZHANG Hai-Yan, XIE Bei-Tao, JIANG Chang-Song, FENG Xiang-Yang, ZHANG Qiao, DONG Shun-Xu, WANG Bao-Qing, ZHANG Li-Ming, QIN Zhen, DUAN Wen-Xue. Screening of leaf physiological characteristics and drought-tolerant indexes of sweetpotato cultivars with drought resistance [J]. Acta Agronomica Sinica, 2022, 48(2): 518-528.
[9] CAO Liang, DU Xin, YU Gao-Bo, JIN Xi-Jun, ZHANG Ming-Cong, REN Chun-Yuan, WANG Meng-Xue, ZHANG Yu-Xian. Regulation of carbon and nitrogen metabolism in leaf of soybean cultivar Suinong 26 at seed-filling stage under drought stress by exogenous melatonin [J]. Acta Agronomica Sinica, 2021, 47(9): 1779-1790.
[10] ZHANG Ming-Cong, HE Song-Yu, QIN Bin, WANG Meng-Xue, JIN Xi-Jun, REN Chun-Yuan, WU Yao-Kun, ZHANG Yu-Xian. Effects of exogenous melatonin on morphology, photosynthetic physiology, and yield of spring soybean variety Suinong 26 under drought stress [J]. Acta Agronomica Sinica, 2021, 47(9): 1791-1805.
[11] YUE Dan-Dan, HAN Bei, Abid Ullah, ZHANG Xian-Long, YANG Xi-Yan. Fungi diversity analysis of rhizosphere under drought conditions in cotton [J]. Acta Agronomica Sinica, 2021, 47(9): 1806-1815.
[12] LI Jie, FU Hui, YAO Xiao-Hua, WU Kun-Lun. Differentially expressed protein analysis of different drought tolerance hulless barley leaves [J]. Acta Agronomica Sinica, 2021, 47(7): 1248-1258.
[13] LI Hui, LI De-Fang, DENG Yong, PAN Gen, CHEN An-Guo, ZHAO Li-Ning, TANG Hui-Juan. Expression analysis of abiotic stress response gene HcWRKY71 in kenaf and transformation of Arabidopsis [J]. Acta Agronomica Sinica, 2021, 47(6): 1090-1099.
[14] LI Peng-Cheng, BI Zhen-Zhen, SUN Chao, QIN Tian-Yuan, LIANG Wen-Jun, WANG Yi-Hao, XU De-Rong, LIU Yu-Hui, ZHANG Jun-Lian, BAI Jiang-Ping. Key genes mining of DNA methylation involved in regulating drought stress response in potato [J]. Acta Agronomica Sinica, 2021, 47(4): 599-612.
[15] ZHAO Jia-Jia, QIAO Ling, WU Bang-Bang, GE Chuan, QIAO Lin-Yi, ZHANG Shu-Wei, YAN Su-Xian, ZHENG Xing-Wei, ZHENG Jun. Seedling root characteristics and drought resistance of wheat in Shanxi province [J]. Acta Agronomica Sinica, 2021, 47(4): 714-727.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!